An Improved Protocol for the Matrigel Duplex Assay: A Method to Measure Retinal Angiogenesis

Neovascular diseases of the retina, such as diabetic retinopathy (DR) and age-related macular degeneration (AMD), are proliferative retinopathies involving the growth of new blood vessels on the retina, which in turn causes impairment and potential loss of vision. A drawback of conventional angiogenesis assays is that they are not representative of the angiogenic processes in the retina. In the retina, the new blood vessels grow (from pre-existing blood vessels) and migrate into a non-perfused region of the eye including the inner limiting membrane of the retina and the vitreous, both of which contribute to vision loss. The Matrigel Duplex Assay (MDA) measures the migration of angiogenic capillaries from a primary Matrigel layer to a secondary Matrigel layer, which resembles the pathological angiogenesis in AMD and DR. The methodology of MDA is comprised of two steps. In the first step, the human retinal microvascular endothelial cells (HRMECs) are mixed with phenol red–containing Matrigel (in a 1:1 ratio) and seeded in the center of an 8-well chamber slide. After 24 h, a second layer of phenol red–free Matrigel is overlaid over the first layer. Over the course of the next 24 h, the HRMECs invade from the primary Matrigel layer to the secondary layer. Subsequently, the angiogenic sprouts are visualized by brightfield phase contrast microscopy and quantified by ImageJ software. The present manuscript measures the angiogenesis-inhibitory activity of the Src kinase inhibitor PP2 in primary HRMECs using the MDA. The MDA may be used for multiple applications like screening anti-angiogenic drugs, measuring the pro-angiogenic activity of growth factors, and elucidating signaling pathways underlying retinal angiogenesis in normal and disease states.


Background
The retina consists of organized layers of photoreceptors, interneurons, glia, epithelial cells, and endothelial cells.The proper maintenance of vascular networks is critical to normal visual function [1].Aberrant angiogenesis is the hallmark of several ocular diseases including age-related macular degeneration (AMD) and diabetic retinopathy (DR) [2].The angiogenic process in the retina is a complex, multistep process involving endothelial cell invasion, adhesion, chemotactic migration, proliferation, and differentiation into capillary tube-like structures and the production of a basement membrane around the vessel [3].A survey of literature shows that the Matrigel capillary tube assay is one of the most prevalent angiogenesis assays in cell culture models [4,5].In this assay, human microvascular endothelial cells are seeded on a three-dimensional layer of solidified Matrigel (or any other reconstituted basement membrane extracellular matrix).Over a period of 24 h, the cells differentiate into capillary tube-like networks, which can be quantified by digital image analysis.A caveat of this assay is that it does not represent pathological angiogenesis in the eye.The Matrigel assay is more representative of vasculogenesis, which is defined as the differentiation of endothelial cells to yield de novo primitive vascular networks rather than angiogenesis where new capillaries are generated from existing vasculature [6].Another disadvantage is the lack of a lumen in the capillaries obtained by this assay [7,8].Other methods used to measure retinal angiogenesis include the measurement of endothelial cell proliferation or endothelial cell chemotaxis or migration.Although these individual processes are useful indicators of angiogenic activity, they do not provide a holistic representation of the angiogenic cascade [7,8].These drawbacks are circumvented by using the Matrigel Duplex Assay (MDA).In the MDA, the angiogenic sprouting (in the secondary Matrigel layer) arises from pre-formed vascular networks in the primary layer [9,10].The MDA represents a combination of all the steps of retinal angiogenesis and provides a highly relevant model for the study of pro-and anti-angiogenic agents in vitro.In-depth morphological studies have shown that the angiogenic sprouting (occurring in the MDA) closely mimics retinal angiogenesis in vivo.Electron microscopy studies have demonstrated the presence of a lumen and elaborate cell-cell junctions within the endothelial cell aggregates observed in the MDA [9,10].As the endothelial capillary tube-like structures invade into the secondary Matrigel layer, the leading edge of the capillary sprouts is associated with long filopodia.The structure of these filopodia resembles those seen at the angiogenic front during developmental angiogenesis in the neonatal retina [9].The retinal endothelial cells grown within the two layers of Matrigel reflect the biological characteristics of the neovascular retina, such as diffusion gradient of oxygen, nutrients, and pH.The growth of the cells inside the Matrigel duplex system allows for complex cell-cell and cell-matrix interaction.Furthermore, these retinal endothelial sprouts may be characterized and quantified by confocal microscopy [9].Stitt et al. (2005) were the first to report the protocol for the MDA to study retinal angiogenesis in diabetic retinopathy [10].During the application of the original protocol of Stitt et al. (2005) in our laboratory, some technical challenges occurred.The original protocol uses phenol red-containing Matrigel for both the primary and the secondary Matrigel layers [9,10].We observed that the interface between the two layers was difficult to visualize by phase contrast microscopy.This is especially true when there is a dense network of retinal capillaries migrating from the primary to the secondary Matrigel layer in the MDA.In order to circumvent this issue, we used a black Sharpie marker pen (in our early studies) to draw a boundary encircling the first layer before adding the second Matrigel layer [11].This approach had its limitations as it was difficult to accurately demarcate the boundary of the first layer.Secondly, the black boundary around the primary layer appeared as a thick black band under the brightfield microscope, increasing the difficulty of accurately visualizing and quantifying the angiogenic capillary sprouts in the second layer.We drew the dotted line in the center of this black band to quantify the in vasion of endothelial capillary networks into the secondary layer [11] and the whole process became arduous and cumbersome.We devised a novel strategy to clearly differentiate between the two Matrigel layers in the MDA by utilizing phenol red-free Matrigel (PR-free-Matrigel) and phenol red-containing Matrigel (PR-Matrigel) for the individual layers.Initially, we tried to use PR-free-Matrigel for the primary layer and PR-Matrigel for the secondary layer but found that this arrangement resulted in poor quality photographs under a microscope.Subsequently, we reversed the layers (using the PR-Matrigel in the primary layer and PR-free-Matrigel in the secondary layer) and obtained clearer pictures under phase contrast microscopy, providing readily reproducible images and assay results.The present manuscript describes the ability of the Src Kinase inhibitor PP2 [12] (at a concentration of 10 µM) to suppress retinal angiogenesis of human retinal microvascular endothelial cells (HRMECs) using our modified MDA protocol.6

EGM-2 media (with growth factors)
We usually make EGM-2 media (with growth factors) in a bottle and add the FBS to the T-75 flask directly.This is because we often do experiments in serum-free conditions, so we add the FBS separately during cell culture as required.

10 mM PP2
Molecular weight of PP2 = 301.78Weigh 6 mg of PP2 in a sterile microcentrifuge tube.In the laminar flow hood, dissolve the PP2 in 2 mL of DMSO.Vortex briefly to obtain 10 mM PP2 stock solution.Aliquot this PP2 stock solution (as 50 µL aliquots) into microcentrifuge tubes and store at -20 °C.6.Return the tissue culture flasks to the humidified tissue culture incubator maintained at 37 °C with 5% CO2.Do not disturb the culture for at least 24 h after the culture has been initiated.After 24 h, aspirate and discard the media in the flask and add fresh EGM-2 complete media to remove any residual DMSO and unattached cells.A healthy culture will display cobblestone morphology and non-granular cytoplasm [9,10].

C. Working with Matrigel
1.The Matrigel is aliquoted in ice-cold sterile Corning freezing vials and stored at -70 °C.2. Before starting the MDA, the required amount of Matrigel should be thawed overnight at 4 °C.The Matrigel is always kept on ice during the experiment.3. Matrigel is a liquid at 4 °C, and it polymerizes into a gel-like solid at 37 °C.We recommend that plasticware and reagents should be kept ice cold while handling the Matrigel.4. The thawed Matrigel is a thick, viscous liquid.Pipette tips should be trimmed at the tip (to increase the diameter of the tip) to transport the Matrigel in/out of the freezing vials.All microcentrifuge tubes and pipette tips should be kept ice cold while handling the Matrigel to avoid polymerization.In our laboratory, we use ice-cold serum-free RPMI-1640 as a cooling media.All pipette tips are dipped into the ice-cool media to chill them down before using them to handle Matrigel.

D. Matrigel Duplex Assay
Day 1 1.The assay is set up in an 8-well chamber slide.Before starting the experiment, draw the schema of the assay (Figure 2).The region (with the checkered pattern) of the right side of the chamber slide is used to lift and transport the slide.with DPBS and transfer the solution to a 15 mL tube.6. Gently spin down the cells at 800× g for 5 min at room temperature in a benchtop centrifuge.Aspirate the media and resuspend the HRMECs in EGM-2 containing 4% FBS. 7. Count the cells using the Corning cell counting chamber.Adjust the concentration of the cells to 1.6 × 10 7 cells/mL (using EGM-2 media containing 4% FBS) in a sterile 5 mL microfuge tube.This tube is labeled as TUBE A. 8.The stock solution of PP2 (10 mM) should be diluted to a concentration of 200 µM (see Recipes) in basal EBM-2 media in a sterile microfuge tube.The PP2 is prepared at 20× working concentration and labeled as PP2-20X.The PP2-20X is the concentration of PP2 used to set up the MDA.The final concentration of PP2 to be used for the MDA is 10 µM. 9.In a separate 1.5 mL microfuge tube, mix the cells (from TUBE A) with PP2-20X at the ratio of 10:1 (v/v).
This dilutes the PP2-20X to a concentration of 20 µM.As an example, you may add 45 µL of cell suspension (from TUBE A) to 5 µL of the PP2-20X.Gently flick the tube so that the drug mixes well with the cell suspension.Do not vortex because this will shear (and lyse) the cells.Therefore, the new concentration of PP2 is 20 µM.This tube is labeled as TUBE B. 10.The vehicle for the PP2-20X is 2% DMSO (hereafter referred to as VEH-20X, see Recipes).Just like in the previous step, mix the cells (from TUBE A) with VEH-20X at the ratio of 10:1 (v/v).Do not vortex because this will shear (and lyse) the cells.Therefore, the new concentration of the vehicle is now 0.2% DMSO.This tube is labeled as TUBE B-VEH.11.Keep a few empty 1.5 mL microfuge tubes on ice to chill them down.Combine the cells with Matrigel at a ratio of 1:1 in an ice-cold microfuge tube.For example, mix 20 µL of Matrigel with 20 µL of cell suspension (taken from TUBE B).Mix the cells with the Matrigel by flicking the tube.Do not allow the tube to warm up and do not generate air bubbles in the Matrigel-cell suspension while flicking.The final PP2 concentration in the tube is now 10 µM.This tube is labeled as TUBE C. 12. Similarly, 20 µL of HRMECs (treated with 0.2% DMSO) are mixed with 20 µL of Matrigel in a separate microfuge tube.The final concentration of vehicle in the tube is now 0.1% DMSO.This tube is labeled as TUBE C-VEH.13.Table 1 describes the tubes A, B, and C in detail.

Tube name Composition of vehicle Contents of tube A
HRMECs resuspended in EGM-2 media supplemented with 4% FBS at a concentration of 1.6 × 10 7 cells/mL.B 45 µL of HRMEC suspension (a concentration of 1.6 × 10 7 cells/ml, taken from TUBE A) mixed with 5 µL of 200 µM PP2 (referred in the text as PP2-20X).Final concentration of PP2 is now 20 µM.Gently flick the tube so that the drug mixes well with the cell suspension.

B-VEH
The vehicle for the PP2- 14.Take a chamber slide out of the packet.Place the chamber slide in a dish over a piece of tissue paper inside the laminar flow hood.Use a Kimwipe or a sterile gauze pad to prevent the chamber slide from sticking to the surface of the hood, as this may cause a disruption of the primary Matrigel layer during the transfer to the incubator.Take 6 µL of the cell suspension from TUBE C and plate it as a drop in the center of the chamber slide.Each sample is assayed in duplicate.A similar process is followed for TUBE C-VEH.The chamber slide is then incubated at 37 °C for 1 h in a humidified cell culture incubator with 5% CO2.Below is a schematic representation of the chamber slide with the drop in the middle of the well (Figure 3).We also provide a photograph of the chamber slide with the drop in the middle of the well (Figure 4).Note: The drop does not need to be exactly at the geometric center of the slide.The important fact to remember is that it should be centered enough so that the cells can invade radially into the secondary layer.
Even if the drop (the primary layer) is slightly off center, that would be okay for the assay.Day 2 1.After 24 h, dilute the PR-free Matrigel with an equal volume of EGM-2 supplemented with 4% FBS.Keep this solution on ice.2. Look at the chamber slide under a phase contrast microscope to confirm the presence of a network of retinal endothelial cell tube-like structures within the primary Matrigel layer (the drop).3. Aspirate the medium from the chamber slide.Use a pipette to gently remove the medium from the cell without disturbing the drop at the center of the chamber slide.4. Gently overlay 200 µL of the 1:1 solution of phenol red-free Matrigel (diluted in EGM-2 supplemented with 4% FBS) over the drop.Figure 6 represents a schematic diagram of the chamber slide.The pink color drop represents the primary layer of Matrigel, and the light grey region (around the pink drop) represents the secondary layer comprised of phenol red-free Matrigel.A side view of the chamber slide is provided in Figure 7.The chamber slide is incubated at 37 °C for 1 h (with 5% CO2) in a cell culture incubator.After 1 h, add 250 µL of EGM-2 complete media to each well.Leave the chamber slide at 37 °C for 24 h in a cell culture incubator.

Day 3
After 24 h, observe the chamber slide under a phase contrast microscope.The angiogenic tube-like structures (from the HRMECs in the primary Matrigel layer) should be clearly seen invading radially into the secondary Matrigel layer.Photograph three independent fields (at 10× magnification) for each sample for quantitative analysis.

E. Processing the images using Adobe Illustrator and ImageJ
1.The figure shown below shows the representative images obtained from the MDA.The black arrows indicate the sprouting angiogenic HRMEC tube-like structures, which have invaded the secondary Matrigel layer from the primary layer (Figure 8).

7 Published 8 Published: Dec 05, 2023 Figure 1 .
Figure 1.Schematic diagram representing the establishment of human retinal microvascular endothelial cell (HRMEC) cultures from cryopreserved cells.The flasks should be placed flat (horizontally) in the cell culture incubator.

9 Published
Cite as: Brown, K.C. et al. (2023).An Improved Protocol for the Matrigel Duplex Assay: A Method to Measure Retinal Angiogenesis.Bio-protocol 13(23): e4899.DOI: 10.21769/BioProtoc.4899.HRMECs: maintenance and subculturing of HRMEC cultures 1. Culture HRMECs to approximately 70%-80% confluence.The media should be replenished once every three days.2. Rehydrate the required number of BioCoat T-75 flasks (see Section A, Step 2). 3. Rinse the cells with DPBS.Use 5 mL of DPBS for T-25 and 10 mL of DPBS for T-75 flasks.4. Add 3 mL of 0.25% Trypsin/EDTA solution into the T-75 flask (in the case of T-25 flask, use 2 mL). 5. Gently rock the flask to ensure that the cells are covered by Trypsin/EDTA solution.Incubate the flask at 37 °C incubator for 1-2 min or until cells are completely rounded up (monitored with inverted microscope).Gently tap the side of the flask to fully detach the cells if necessary.6. Add 2 mL of FBS to the T-75 flask (use 1 mL for a T-25 flask) to neutralize the Trypsin-EDTA.Using a 5 mL serological pipette, gently rinse the flask completely and transfer the cells in a 15 mL centrifuge tube.7. Rinse the growth surface of the flask with 10 mL of DPBS to collect the remainder of the detached cells and combine in the same 15 mL centrifuge tube from Step B6.Close and examine the flask under an inverted microscope to verify that the cell harvesting is complete.There should be less than 5% cells (of the initial density of the flask) remaining in the flask.8. Centrifuge the harvested cell suspension at 800× g for 5 min at room temperature using a benchtop centrifuge.Remove (aspirate) the FBS/DPBS liquid from the pellet.Gently flick the tube to dislodge the cells and resuspend them in 1 mL of EGM-2 complete medium.9. Count the cells using a hemocytometer and then dispense the required volume of cell suspension into the hydrated T-75 flask as recommended in see Section A, Steps 5-6.Return the flasks back to the cell culture incubator maintained at 37 °C and 5% CO2.

11 Published
20X is 2% DMSO (referred in the text as VEH-20X) 45 µL of HRMEC suspension (a concentration of 1.6 × 10 7 cells/mL, taken from TUBE A) and mixed with 5 µL of 2% DMSO (which is the vehicle for PP2-20× and also called VEH-20% in the text).C Mixture of 20 µL of Matrigel with 20 µL of cell suspension (treated with PP2 and taken from TUBE B).Mix the cells with the Matrigel by flicking the tube.C-VEH TUBE C-VEH contains a mixture of 20 µL of Matrigel with 20 µL of cell suspension (treated with vehicle and taken from TUBE B-VEH).Mix the cells with the Matrigel by flicking the tube.Cite as: Brown, K.C. et al. (2023).An Improved Protocol for the Matrigel Duplex Assay: A Method to Measure Retinal Angiogenesis.Bio-protocol 13(23): e4899.DOI: 10.21769/BioProtoc.4899.

Figure 3 .
Figure 3. Schematic diagram of the chamber slide with the drop in the middle of the well

Figure 4 .
Figure 4. Photograph of the chamber slide with the primary layer in the middle of the slide

Figure 5 .
Figure 5. Schematic diagram of the side view of the chamber slide.(A) The components of the chamber slide have been labeled.(B) The of adding the primary layer (PR-containing Matrigel and HRMECs) in the center of the chamber slide.(C) The drop is allowed to polymerize for 1 h at 37 °C (with 5% CO2) in a cell culture incubator.(D) After 1 h, 250 µL of EGM-2 complete media is added to each well.

Figure 6 .Figure 7 .
Figure 6.Schematic diagram of the chamber slide with both the primary and secondary layers

Published: Dec 05, 2023 Figure 8 .
Figure 8. Representative photographs obtained from the Matrigel Duplex Assay (MDA).The black arrows indicate the angiogenic tube-like structures invading into the secondary layer.

Figure 9 .
Figure 9. Drawing the dotted line at the interface of the two layers using Adobe Illustrator 2023

Figure 10 .
Figure 10.Representative image of the Matrigel Duplex Assay (MDA) with lines drawn using Adobe Illustrator

Figure 11 .
Figure 11.Microscope images were analyzed by the ImageJ program (by three independent observers).Data were graphically represented using GraphPad Prism (Version 9).Data were analyzed through an unpaired non-parametric t-test followed by the Mann-Whitney test.Values represented by the same letter are not statistically significantly different from each other (P ≤ 0.05).
Thaw out one aliquot of PP2 (concentration = 10 mM) in a water bath (held at 37 °C).Add 2 µL of PP2 in 100 µL of basal EBM-2 medium.Vortex vigorously.Now the concentration of PP2 is 200 µM (called PP2 -20X in the text).Use this PP2 solution as described in Section D, Step 8. Discard the remaining solution of PP2-20X.